Interaction of Escherichia coli DNA polymerase I (Klenow fragment) with primer-templates containing N-acetyl-2-aminofluorene or N-2-aminofluorene adducts in the active site

Abstract

DNA adducts formed by aromatic amines such as N-acetyl-2-aminofluorene (AAF) and N-2-aminofluorene (AF) are known to cause mutations by interfering with the process of DNA replication. To Understand this phenomenon better, a gel retardation assay was used to measure the equilibrium dissociation constants for the binding of an exonuclease-deficient Escherichia coli DNA polymerase I (Klenow fragment) to DNA primer-templates modified with an AAF or AF adduct. The results indicate that the nature of the adduct as well as the presence and nature of an added dNTP have a significant influence on the strength of the binding of the polymerase to the DNA. More specifically, it was found that the binding is 5-10-fold stronger when an AAF adduct, but not an AF adduct, is positioned in the enzyme active site. In addition, the polymerase was found to bind the unmodified primer-template less strongly in the presence of a noncomplementary dNTP than in the presence of the correct nucleotide. The same trend holds true for the primer-template having an AF adduct, although the magnitude of this difference was lower. In the case of the AAF adduct, the interaction of the polymerase with the primer-template was stronger and almost independent of the nucleotide present.